Electrochemical biosensor of light-driven nanopore channel and application of electrochemical biosensor

Abstract

The invention discloses an electrochemical biosensor of a light-driven nanopore channel and application of the electrochemical biosensor. According to the sensor, a photoresponse nanopore membrane is constructed, an outer surface aptamer probe is combined, asymmetric photothermal gradient driven ion transmission induced by near-infrared light is utilized, and ultra-sensitive detection on a target object is realized by regulating and controlling the charge density of the outer surface. According to the electrochemical biosensor provided by the invention, traditional voltage driving is replaced by asymmetric near-infrared light driving, so that the electrochemical biosensor has a relatively wide detection range and an extremely low detection limit, and high-sensitivity and specific detection on a target object can be realized. The electrochemical biosensor can be used for detecting microcystin-LR (MC-LR), the detection limit reaches 1 * 10 <-7 > mu g / L, the sensitivity is improved by 5 orders of magnitude compared with that of a traditional voltage-driven sensor, and the electrochemical biosensor can be applied to trace pollutant detection in the fields of environmental monitoring, food safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to an electrochemical biosensor with light-driven nanopores and its applications. Background Technology

[0002] Aptamer sensors based on nanopores have become a research hotspot due to their advantages such as high sensitivity, label-free operation, and rapid response. Traditional voltage-driven nanopore sensors rely on steric hindrance changes (such as conformational rearrangements) induced by aptamer-target binding to affect transmembrane ion currents. However, the aptamers require cumbersome structural optimization to generate detectable signals, limiting the universality and sensitivity of this method. Therefore, developing novel nanopore sensing strategies with high sensitivity that do not require significant conformational changes in aptamers is of great significance. Summary of the Invention

[0003] The purpose of this invention is to address the problems of traditional voltage-driven nanopore sensors, which rely on aptamer conformational changes and have insufficient sensitivity, by providing an electrochemical biosensor with light-driven nanopores and its application. This sensor achieves ultrasensitive detection without significant aptamer conformational changes by inducing an asymmetric temperature gradient through photothermal effects to regulate the charge density on the outer surface.

[0004] The primary objective of this invention is to provide a light-driven nanoporous electrochemical biosensor, comprising: a light-responsive nanoporous film, an aptamer probe, and a near-infrared light source; the light-responsive nanoporous film is formed by assembling nanosheets layer by layer, with sub-nanometer spacing between adjacent monolayer nanosheets, constituting sub-nanometer ion channels; the aptamer probe is modified on the outer surface of the light-responsive nanoporous film for specific recognition of target molecules; under asymmetric near-infrared light irradiation, the light-responsive nanoporous film exhibits a temperature gradient through photothermal conversion, thereby promoting the binding of the aptamer probe with the target molecule and causing a change in the charge density on the outer surface of the film, thus achieving the detection of the target molecule.

[0005] Furthermore, the photoresponsive nanoporous film comprises Ti3C2T X Any one of MXene membrane, graphene oxide membrane, carbon nanotube membrane, or porous ionomer membrane with photothermal conversion.

[0006] Furthermore, the photoresponsive nanoporous film is Ti3C2T X MXene membrane.

[0007] Furthermore, the method for preparing the photoresponsive nanoporous thin film includes the following steps: S11. Using HCl / LiF as the etching solution, add the MAX phase Ti3AlC2 and heat and stir the mixture for a period of time. S12. The etching product obtained in step S11 is centrifuged and washed with HCl and deionized water. After the pH value of the supernatant reaches neutral, the supernatant is ultrasonically treated in an inert environment for a period of time. After centrifugation again, the supernatant is taken as the MXene nanosheet colloidal solution. S13. The MXene nanosheet colloidal solution obtained in step S12 is vacuum filtered through a mixed cellulose filter membrane, and then the product is vacuum dried to obtain a photoresponsive nanoporous film.

[0008] Furthermore, the volume-to-mass ratio of HCl to LiF in the HCl / LiF etching solution is 80 mL: (7~8) g, and the mass ratio of Ti3AlC2 to LiF is 1: (2~3); the heating temperature is 40~50℃, and the heating time is 36~48 h.

[0009] Furthermore, the method for modifying the aptamer probe on the outer surface of the photoresponsive nanoporous film specifically includes the following steps: S21. On one side surface of the nanoporous membrane obtained in step S13, a chromium layer and a gold layer are deposited sequentially using a vapor deposition method. S22. The aptamer probe solution is uniformly coated onto the surface of the deposited gold layer and incubated. S23. Wash the unbound aptamer probe with Tris-HCl buffer and air dry at room temperature to obtain the electrochemical biosensor.

[0010] Furthermore, the thickness of the chromium layer is 1~3 nm, and the thickness of the gold layer is 5~10 nm.

[0011] A second objective of this invention is to provide the application of the above-described electrochemical biosensor in the detection of target molecules.

[0012] Furthermore, the electrochemical biosensor is used for qualitative and quantitative detection of microcystin-LR.

[0013] A third objective of the present invention is to provide a target molecule detection system, including the above-described electrochemical biosensor.

[0014] Furthermore, the electrochemical biosensor is placed in a dual-cell electrolytic cell, which is divided into two chambers. Ag / AgCl electrodes are inserted into the two chambers respectively, and KCl solution is added. Near-infrared light is used to irradiate the electrochemical biosensor through the electrolytic cell.

[0015] Furthermore, the concentration of the KCl solution is 10. -5 ~10 -1 M.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The photo-driven nanoporous electrochemical biosensor constructed in this invention significantly improves sensor performance by utilizing the synergistic effect of the photothermal properties of the photoresponsive nanoporous film and the regulation of the external surface charge. Based on the photothermal conversion capability, high specific surface area layered structure, and adaptable in vitro surface directional modification of the photoresponsive nanoporous film, this sensor utilizes near-infrared light-induced asymmetric photothermal gradient to drive ion transport. By regulating the external surface charge density, it achieves ultrasensitive detection of the target analyte, effectively reducing ion transport resistance and increasing target recognition sites. This enables highly sensitive and specific detection of trace MC-LR with a wide detection range (1×10⁻⁶). -7 ~1×10 -3 μg / L, low detection limit (LOD as low as 1×10 μg / L), and low detection limit (LOD as low as 1×10 μg / L). -7 μg / L).

[0017] (2) Compared with traditional voltage-driven nanochannel sensors, the light-driven nanochannel sensor of the present invention has significant advantages: signal generation does not depend on significant conformational changes of the aptamer, and current response can be achieved only by a small adjustment of the charge density on the outer surface; near-infrared light replaces electric field driving, simplifying the complexity of the detection equipment, and the light response speed is fast, enabling rapid acquisition of detection results. The present invention is of great significance for breaking through the sensitivity bottleneck of traditional nanochannel sensors and promoting the industrial application of rapid on-site detection technology for trace pollutants in the fields of environmental monitoring and food safety.

[0018] (3) The application of the electrochemical biosensor of the present invention is based on the ion-selective transport mechanism regulated by photothermal effect to improve sensitivity. Its core principle is as follows: In order to achieve high-sensitivity detection under light-driven conditions, the sensor is composed of a layered photoresponsive nanoporous membrane and an aptamer probe fixed on the outer surface. The detection of MC-LR is achieved based on the ion-selective change regulated by photothermal effect; by utilizing the efficient photothermal conversion characteristics of the photoresponsive nanoporous membrane under near-infrared light irradiation, near-infrared light is irradiated on one side of the nanoporous membrane to establish an asymmetric temperature gradient to drive ion transport, replacing the traditional voltage-driven method; in this process, the negatively charged nanoporous interface preferentially selects cations (such as K+) through charge interaction. + When the aptamer probe on the outer surface captures MC-LR, the charge density on the outer surface of the nanopore changes, enhancing the adsorption and transport capacity for cations, thereby amplifying the transmembrane ion current signal. This detection principle breaks through the limitation of traditional sensors that rely on aptamer conformational rearrangement. It only requires a slight change in the charge density on the outer surface to achieve signal conversion. It can be adapted to any aptamer probe, greatly expanding the range of detectable targets.

[0019] (4) The electrochemical biosensor with light-driven nanopores provided by the present invention has a simple fabrication process, controllable cost, good stability and repeatability, and has the potential for market application. Attached Figure Description

[0020] Figure 1a Here is an atomic force microscope image of the MXene single nanosheets prepared in the embodiments of the present invention; Figure 1b This is a height analysis diagram of the MXene single nanosheets prepared in the embodiments of the present invention; Figure 2 This is a stability diagram of the MXene nanoporous membrane prepared in the embodiments of the present invention in water; Figure 3a This is a scanning electron microscope image of the surface of the MXene nanoporous membrane prepared in Example 2 of the present invention; Figure 3b This is a scanning electron microscope image of the cross-section of the MXene nanoporous membrane prepared in Example 2 of the present invention; Figure 4 X-ray diffraction patterns of the MXene nanoporous membrane, the aptamer probe-modified MXene nanoporous membrane (APT@MXene), and the MXene nanoporous membrane (APT@MXene + MC-LR) after MC-LR capture in the embodiments of the present invention. Figure 5 The images show the X-ray photoelectron spectra of the APT@MXene film, the APT@MXene film, and the MXene film after argon ion sputtering in the embodiments of the present invention. Figures 6a-6b These are schematic diagrams of the target molecule detection system constructed in the embodiments of the present invention and the test device of the traditional voltage-driven MXene nanopore aptamer electrochemical biosensor in Comparative Example 1. Figure 7 The APT@MXene film binding concentration in Examples 1-5 of this invention is 1×10⁻⁶. -3 Current-time curves and specific current growth rates of μg / L MC-LR before and after near-infrared light driving; Figure 8a In Example 2 of this invention, the MXene film and APT@MXene film are combined with MC-LR (1×10⁻⁶). -3 Current-time curves before and after (μg / L) under near-infrared light drive; Figure 8b In the comparative example of this invention, MXene film and APT@MXene film are combined with MC-LR (1×10). -3 Voltage-current curves before and after (μg / L) under voltage drive; Figure 9 This is a standard concentration-rate of change curve for the light-driven MXene nanopore sensor in Embodiment 2 of the present invention and the comparative conventional voltage-driven nanopore sensor. Figure 10a Example 2 of the present invention is a light-driven MXene nanopore sensor. Figure 6a ) Specificity detection diagram for MC-LR; Figure 10b To drive the MXene nanopore sensor with a comparative voltage ( Figure 6b ) Specificity detection diagram for MC-LR; Figure 11 This is a dynamic curve showing the continuous and synchronous changes in photothermal voltage (ΔV) and temperature change (ΔT) recorded when the light-driven MXene nanopore sensor in Embodiment 2 of the present invention undergoes five temperature cycles. Figure 12a The image shows the zeta potential of the MXene film prepared in this embodiment of the invention, the aptamer probe used, and MC-LR in 0.1 M KCl solution. Figure 12b The surface charge density of the MXene film, APT@MXene, and APT@MXene+MC-LR prepared in Example 2 of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] This invention provides an electrochemical biosensor with light-driven nanopores, comprising a light-responsive nanopore film, an aptamer probe, and a near-infrared light source. The light-responsive nanopore film is formed by assembling nanosheets layer by layer, with sub-nanometer spacing between adjacent monolayer nanosheets, constituting sub-nanometer ion channels. The aptamer probe is modified on the outer surface of the light-responsive nanopore film for specific recognition of target molecules. Under asymmetric near-infrared light irradiation, the light-responsive nanopore film exhibits a temperature gradient through photothermal conversion, thereby promoting the binding of the aptamer probe with the target molecule and causing a change in the charge density on the outer surface of the film, thus achieving the detection of the target molecule.

[0023] In some embodiments, the above-mentioned photoresponsive nanoporous thin film includes Ti3C2T XAny one of MXene films, graphene oxide films, carbon nanotube films, or porous ionomer films with photothermal conversion. The above-mentioned photoresponsive nanoporous films can be prepared using methods described in references or conventional techniques in the art. Graphene oxide films themselves possess good hydrophilicity and photothermal properties; through hydration, 2D nanochannels can be formed between their layers. Under light irradiation, especially after partial reduction or composite with other materials (to obtain rGO), their photothermal effect is significantly enhanced. Carbon nanotube films (single-walled or multi-walled) exhibit strong absorption and excellent photothermal properties over a broad spectral range (especially the near-infrared region). They can be prepared as independent films or deposited on other porous substrates (such as anodic aluminum oxide, AAO) to form composite films. Ti3C2T X MXene membranes, being a novel class of two-dimensional metal carbides / nitrides, exhibit excellent photothermal conversion performance due to their high electrical conductivity and metallic properties; the channels formed by their layered stacking are highly ordered. Porous ionomer membranes with photothermal conversion capabilities are functional membrane materials that organically combine porous structures, ionic functional groups, and a polymer matrix. Through functionalized composite strategies, highly efficient photothermal nanomaterials can be introduced into the matrix, pores, or surface of porous ionomer membranes, thereby preparing novel photothermal responsive porous ionomer membranes.

[0024] Utilizing the high-efficiency photothermal conversion characteristics of the aforementioned photoresponsive nanoporous films under near-infrared light irradiation, near-infrared light is irradiated on one side of the nanoporous film to establish an asymmetric temperature gradient to drive ion transport, replacing the traditional voltage-driven method. When the adapted probe on the outer surface of the photoresponsive nanoporous film captures target molecules, the charge density on the outer surface of the nanopores changes, enhancing the adsorption and transport capacity for cations, thereby amplifying the transmembrane ion current signal and realizing the detection of target molecules.

[0025] This invention provides an example of a light-driven nanoporous thin film selected from Ti3C2T. X The preparation method of the MXene membrane includes the following steps: Step S11: Using HCl / LiF as the etching solution, add MAX phase Ti3AlC2, and heat and stir the mixture for a period of time; Step S12: The etching product obtained in step S11 is centrifuged and washed with HCl and deionized water. After the pH value of the supernatant reaches neutral, the supernatant is ultrasonically treated in a nitrogen environment for a period of time. After centrifugation again, the supernatant is taken as the MXene nanosheet colloidal solution. Step S13: The MXene nanosheet colloidal solution obtained in step S12 is vacuum filtered through a mixed cellulose filter membrane, and then the product is vacuum dried to obtain an MXene nanoporous membrane.

[0026] The method for modifying aptamer probe molecules on the outer surface of the MXene nanoporous film includes the following steps: Step S21: On one side surface of the nanoporous membrane obtained in step S13, a chromium layer and a gold layer are deposited sequentially using a vapor deposition method. The thickness of the metallic chromium is 1~3 nm and the thickness of the metallic gold is 5~10 nm. Step S22: Coat the microcystin-LR (MC-LR) aptamer probe solution uniformly onto the surface of the deposited gold layer and incubate at room temperature for 10-12 h; Step S23: Wash the unbound probe with Tris-HCl buffer, air dry at room temperature to obtain APT@MXene nanoporous film.

[0027] In some embodiments, the HCl / LiF etching solution in step S11 is a mixed solution of HCl (mass fraction of 36%) and LiF with a volume-to-mass ratio of 80:7~8 (mL:g), a mass ratio of Ti3AlC2 to LiF of 1:(2~3), an etching temperature of 40~50 ℃, and an etching time of 36~48 h.

[0028] In some embodiments, the centrifugal washing process in step S12 involves washing 3-5 times with 1 M HCl, followed by washing with deionized water until the pH of the supernatant reaches neutral. The mixture is then sonicated under nitrogen for 20-40 minutes, and centrifuged at 3000-4000 r / min for 20-40 minutes.

[0029] In some embodiments, the concentration of the MXene nanosheet colloidal solution in step S13 is 0.5~1 mg / mL, and it is vacuum filtered using a mixed cellulose filter membrane with a pore size of 0.22 μm and a diameter of 50 mm. The filtered product is then vacuum dried at 50~60°C for 6-8 h.

[0030] In some embodiments, the concentration of the aptamer probe solution in step S22 is 0.5~2 μM, and the solvent is Tris-HCl buffer solution.

[0031] Another objective of this invention is to provide the application of the above-described electrochemical biosensor in the detection of target molecules.

[0032] In some embodiments, the electrochemical biosensor of the present invention is used for qualitative and quantitative detection of microcystin-LR. The CAS number for microcystin-LR is 101043-37-2.

[0033] Another object of the present invention is to provide a target molecule detection system comprising the electrochemical biosensor of the present invention. The electrochemical biosensor is disposed in a dual-cell electrolytic cell, which is divided into two chambers. Ag / AgCl electrodes are inserted into each of the two chambers, and KCl solution is added. Near-infrared light is then used to irradiate the electrochemical biosensor through the electrolytic cell.

[0034] In some implementations, the KCl solution concentration can be 10. -5 -10 -1 M.

[0035] The specific process for detecting microcystin-LR using the target molecule detection system provided by this invention is as follows: Step S1: Sample preparation 50-200 μL of MC-LR solution is uniformly coated on the surface of the APT@MXene film obtained in step S23. After incubation at room temperature for more than 2 hours, the film is washed with deionized water 3-5 times and air-dried at room temperature to obtain the APT@MXene film that recognizes and captures MC-LR, denoted as APT@MXene + MC-LR. To obtain a standard curve for detecting MC-LR, MC-LR solutions of different concentrations are prepared with deionized water to prepare APT@MXene films that recognize different concentrations of MC-LR. Step S2, Target Molecular Detection System The obtained APT@MXene + MC-LR film was fixed in the center of a dual-cell electrolytic cell. An equal volume of KCl solution was added to the cell, and an Ag / AgCl electrode was inserted. The nanoporous membrane was irradiated with near-infrared light (808 nm, 100-200 mW) through the electrolytic cell, with unilateral irradiation maintained during the test. Step S3: Ion current signal detection The transmembrane ion current was measured after connecting a picoammeter to an Ag / AgCl electrode. The parameters were set as follows: mode was IT test, time was 1 min, voltage was 0V, and the electrode used was an Ag / AgCl electrode.

[0036] The ion current signal of the APT@MXene thin film is denoted as... The ion current signals of APT@MXene films that identify different concentrations of MC-LR are denoted as... The rate of change of transmembrane ion current ,in i Table of MC-LR solutions of different concentrations.

[0037] Step S4: Draw the standard curve With the concentration of MC-LR solution as the x-axis and the rate of change of transmembrane ion current as the y-axis, a standard curve was plotted by fitting the curve using a logistic regression model.

[0038] The electrochemical biosensor of this invention enables highly sensitive and specific detection of trace MC-LR with a wide detection range (1×10⁻⁶). -7 -1×10 -3 μg / L, low detection limit (LOD as low as 1×10 μg / L), and low detection limit (LOD as low as 1×10 μg / L). -7 μg / L).

[0039] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes them in conjunction with the data, charts and other information of the experimental process.

[0040] The base sequence of the aptamer DNA molecule used in the embodiments of the present invention is 5′-HS-C6-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3′.

[0041] Example 1 This embodiment uses Ti3C2T X MXene membranes are used to construct electrochemical biosensors for detecting MC-LR molecules using photoresponsive nanoporous films.

[0042] S1. Preparation of photoresponsive nanoporous thin films S11. Add 0.7 g Ti3AlC2 to the HCl / LiF etching solution, which is a mixture of 1.4 g LiF and 16 mL HCl (mass fraction 36%). Heat the dispersion to 45 °C and stir for 2 days. S12. Wash the etching product obtained in step S11 by centrifugation with 1 M HCl 3-5 times (10000 r / min, 1 min each time), then wash with water until the pH of the supernatant reaches neutral. Sonicate the supernatant in a nitrogen atmosphere for 20-40 min, and centrifuge at 3500 r / min for 20-40 min. The supernatant is the MXene nanosheet colloidal solution. S13. Dilute the MXene nanosheet colloidal solution obtained in step S12 to 1 mg / mL, and sonicate 40 mL of the solution for 10 minutes to ensure uniform dispersion. Vacuum filter the MXene nanosheet colloidal solution using a mixed cellulose membrane with a pore size of 0.22 μm and a diameter of 50 mm. Then, vacuum dry the product at 50-60 °C for 6-8 h to obtain a photoresponsive nanoporous film, labeled as MXene nanoporous film.

[0043] S2. Modifying aptamer probes on the outer surface of photoresponsive nanoporous films Step S21: On one side surface of the nanoporous membrane obtained in step S13, a chromium layer and a gold layer are deposited sequentially using a vapor deposition method. The thickness of the metallic chromium is 1-3 nm, preferably 2 nm, and the thickness of the metallic gold is 5-10 nm, preferably 10 nm. Step S22: Dilute the MC-LR aptamer to 1 μM using Tris-HCl buffer solution. Take 50-200 μL of the diluted aptamer solution and evenly spread it on the surface of the gold deposited film, and incubate at room temperature for 10-12 h; Step S23: Wash the unbound probe 2-3 times with Tris-HCl buffer, and air dry at room temperature to obtain the APT@MXene nanoporous film.

[0044] After identifying the target using the APT@MXene nanoporous film of this embodiment, it is combined with a near-infrared light source, a transparent dual-cell electrolytic cell, KCl solution, and Ag / AgCl electrode to form a light-driven MXene nanoporous aptamer electrochemical biosensor for detecting MC-LR molecules. The concentration of the MC-LR solution is 1×10⁻⁶. -7 1×10 -6 1×10 -5 1×10 -4 1×10 -3 μg / L, with deionized water as the solvent.

[0045] The steps for testing the MC-LR solution are as follows: (1) Sample preparation 50-200 μL of MC-LR solution (1×10⁻⁶) of different concentrations was uniformly coated on the surface of the APT@MXene film obtained in step S23. -7 1×10 -6 1×10 -5 1×10 -4 1×10 -3 (μg / L), incubate at room temperature for more than 2 hours, wash with deionized water 3-5 times, and air dry at room temperature to obtain the APT@MXene film that recognizes and captures MC-LR, denoted as APT@MXene + MC-LR; (2) Assembly of target molecule detection system The obtained APT@MXene + MC-LR film was immobilized in the center of a dual-cell electrolytic cell, and an equal volume of KCl solution with a concentration of 1×10⁻⁶ was added to the cell. -5 M was inserted, and an Ag / AgCl electrode was inserted. The nanoporous membrane was irradiated through the electrolytic cell using near-infrared light (808 nm, 100-200 mW), with unilateral irradiation maintained during the test. (3) Ion current signal detection The transmembrane ion current was measured after connecting a picoammeter to an Ag / AgCl electrode. The parameters were set as follows: mode was IT test, time was 1 min, voltage was 0V, and the electrode used was an Ag / AgCl electrode.

[0046] The ion current signal of the APT@MXene thin film is denoted as... The ion current signals of APT@MXene films that identify different concentrations of MC-LR are denoted as... The rate of change of transmembrane ion current ,in i Table of MC-LR solutions of different concentrations.

[0047] (4) Draw the standard curve With the concentration of MC-LR solution as the x-axis and the rate of change of transmembrane ion current as the y-axis, a standard curve was plotted by fitting the curve using a logistic regression model.

[0048] Example 2 The operation is the same as in Example 1, except that the concentration of the KCl solution added during sensor assembly is 1×10⁻⁶. -4 M.

[0049] Example 3 The operation is the same as in Example 1, except that the concentration of the KCl solution added during sensor assembly is 1×10⁻⁶. -3 M.

[0050] Example 4 The operation is the same as in Example 1, except that the concentration of the KCl solution added during sensor assembly is 1×10⁻⁶. -2 M.

[0051] Example 5 The operation is the same as in Example 1, except that the concentration of the KCl solution added during sensor assembly is 1×10⁻⁶. -1 M.

[0052] Comparative Example 1 Example 1 was repeated using the same steps S1 and S2, except that the voltage-driven MXene nanopore aptamer electrochemical biosensor does not require near-infrared light irradiation. Specifically, in steps (2) and (3) of the detection process, after fixing the membrane at the center of the dual-electrode ion transmembrane current testing device, the transmembrane ion current was directly tested using a picoammeter without near-infrared light irradiation. The picoammeter parameters were set as follows: mode IV test, voltage range -2V to 2V, number of cycles 5; electrode Ag / AgCl electrode.

[0053] To better illustrate the high detection sensitivity and specificity of the light-driven MXene nanopore aptamer electrochemical biosensor of the present invention for detecting MC-LR, the applicant conducted the following research: like Figure 1a and Figure 1b The figures shown are the AFM images and height analysis diagrams of MXene single nanosheets etched with etching solution in an embodiment of the present invention. Figure 1a As can be seen from the figure, the lateral size of the MXene prepared in the example is 200-500 nm; according to the height analysis diagram, the thickness of the MXene single nanosheet is relatively uniform, with an average thickness of about 2 nm.

[0054] like Figure 2 The figure shows the stability of the MXene nanoporous membrane prepared in this embodiment of the invention after 7 days in water. The figure shows that after 1 day, 2 days, and 7 days, the MXene nanoporous membrane prepared in this embodiment did not disperse, proving that the MXene nanoporous membrane prepared in this embodiment has good stability in aqueous solution.

[0055] like Figure 3a The image shown is a scanning electron microscope (SEM) image of the MXene nanoporous membrane prepared in an embodiment of the present invention. Figure 3b The image shows a cross-sectional scanning electron microscope (SEM) image. The SEM image reveals that the MXene film has a stacked layered microstructure. The gaps between adjacent two-dimensional nanosheets form sub-nanometer-scale ion transport channels, proving that the MXene nanoporous membrane was successfully synthesized.

[0056] like Figure 4 The image shows the X-ray diffraction spectra of the MXene nanoporous film, APT@MXene film, and APT@MXene+MC-LR film in the embodiments of the present invention. A distinct (002) diffraction peak can be observed at 6.22° for all three films. The interlayer spacing is calculated to be 14.22 Å using the Bragg equation. The interlayer spacing of MXene, APT@MXene, and APT@MXene+MC-LR remains consistent, indicating that the external surface modification did not affect the pore size of the nanopores.

[0057] Figure 5 The images show the X-ray photoelectron spectra of the APT@MXene film, the APT@MXene film, and the MXene film with deposited gold layer after argon ion etching, as described in the embodiments of the present invention. Figure 5As shown, the characteristic peak positions of O and C elements in the APT@MXene film are consistent with those of the original MXene film, proving that the aptamer probe grafting modification did not change the film surface structure. The newly added N1s (binding energy approximately 399.8 eV) and P2p (binding energy approximately 133.9 eV) characteristic peaks can be attributed to the introduction of nitrogen-containing bases and the phosphate backbone in the aptamer probe. Further argon ion etching caused the N1s and P2p characteristic peaks to disappear, while the Ti2p and F1s characteristic signals of the original MXene film reappeared. These phenomena indicate that the aptamer probe was successfully and selectively modified on the outer surface of the MXene nanoporous film without penetrating into the film interior, and can be effectively removed by argon ion sputtering etching.

[0058] like Figures 6a-6b The figures shown are schematic diagrams of the target molecule detection system constructed in the embodiments of the present invention and the test device of the traditional voltage-driven MXene nanopore aptamer electrochemical biosensor in Comparative Example 1.

[0059] like Figure 7 The figures show the current-time curves and specific current growth rates of the APT@MXene films in Examples 1-5 of this invention before and after combining with MC-LR under near-infrared light. It can be seen from the figures that after turning on the near-infrared light, the transmembrane ion current of the sensors in each embodiment after combining with MC-LR increases compared to before combination. The current change rate of the sensors constructed in Examples 3-5 is less than the minimum threshold for effective detection (current change rate <20%). The current change rate of Examples 1-2 is significantly higher than the other three examples, and Example 2 uses 1×10... -4 When KCl is used as the electrolyte, the rate of change of ion current is the highest.

[0060] like Figures 8a-8b As shown, these are MXene, APT@MXene, and APT@MXene + MC-LR (MC-LR concentration is 1×10⁻⁶) prepared in Example 2 of this invention, respectively. -3 It curves of three light-driven nanopore sensors (μg / L), and the prepared MXene, APT@MXene, and APT@MXene + MC-LR (MC-LR concentration of 1×10 μg / L) in Comparative Example 1. -3 The IV curves of three voltage-driven nanoporous sensors (μg / L) are shown in Figure 8a. As can be seen from Figure 8a, when the near-infrared light is turned on, the transmembrane current rises rapidly. All three nanoporous membranes exhibit fast photoresponse speeds. The maximum current of the MXene nanoporous membrane is 29.8 ± 0.48 nA, while the current of the APT@MXene film drops to 15.4 ± 0.59 nA, compared to a concentration of 1×10⁻⁶ μg / L. -3After binding with μg / L MC-LR, the transmembrane ion current recovered to 27.9 ± 0.77 nA. From Figure 8b As can be seen, the ion current of the three voltage-driven nanopore sensors increases linearly with the increase of the applied voltage, but the difference in current value is minimal after combining with MC-LR. This proves that the MXene nanopore sensor of this invention has a significantly higher detection capability for MC-LR than the traditional voltage-driven system.

[0061] like Figure 9 The figures show the dynamic range of the ion current change rate under different gradient standard concentrations of MC-LR (known concentration solutions), respectively, for the light-driven MXene nanopore aptamer sensor prepared in Example 2 and the voltage-driven MXene nanopore aptamer sensor prepared in Comparative Example 1; standard curves for the detection of MC-LR by the two sensors were determined. It can be seen from the figures that as the MC-LR concentration increases from 1×10⁻⁶, the ion current changes over time. -7 Increase μg / L to 1×10 -3 In Example 2, the rate of change of transmembrane ion current in the light-driven MXene nanopore aptamer sensor significantly changed from 21.25% to 81.13% at a concentration of μg / L. Based on a 20% rate of change threshold, the limit of detection (LOD) for MC-LR by this aptamer sensor was determined to be 1 × 10 μg / L. -7 μg / L, a value seven orders of magnitude lower than the World Health Organization (WHO) guidance. In contrast, the LOD of the voltage-driven MXene nanopore aptamer sensor in Comparative Example 1 for detecting MC-LR was only 1 × 10⁻⁶. -2 The sensitivity of the MC-LR sensor is 100,000 times lower than that of the light-driven MXene nanopore aptamer sensor, which has a concentration of μg / L.

[0062] like Figures 10a-10b As shown, these are the light-driven MXene nanopore sensor pairs from Embodiment 2 of the present invention. Figure 10a ) and comparative voltage-driven MXene nanopore sensor ( Figure 6b The specificity detection diagram for MC-LR is shown. Besides MC-LR, three other microcystin variants have been found in surface water and drinking water sources: MC-RR, MC-YR, and MC-L. These three variants were selected as interferants. Figure 10a As shown, when the light-driven MXene nanopore sensor in Example 2 detects a concentration of 1×10⁴ for these four MC analogs... -4 At μg / L, the corresponding photoinduced current rise rates were 63.33%, 3.00%, 10.67%, and 4.67%, respectively. Statistical significance analysis showed a P-value less than 0.001, confirming that the adapted sensor has good specificity for MC-LR detection. In contrast, such as Figure 10bAs shown, within the same concentration range, the voltage-driven MXene nanopore sensor in Comparative Example 1 exhibits extremely low specificity, demonstrating that the optically driven MXene nanopore sensor of this invention is also superior to the traditional voltage-driven MXene nanopore sensor in terms of specificity.

[0063] like Figure 11 The figure shows the dynamic curves of the continuous and synchronous changes in photothermal voltage (ΔV) and temperature change (ΔT) recorded during five temperature cycles of the light-driven MXene nanopore sensor in Embodiment 2 of the present invention. As can be seen from the figure, under local near-infrared light irradiation, significant increases in temperature and open-circuit voltage were observed in both the MXene film and the APT@MXene film before and after MC-LR bonding. After the irradiation stopped, ΔT and ΔV gradually decreased synchronously over time. Given the inherent photothermal effect of MXene and the constant 808nm laser power of 150 mW, the photoinduced temperature gradient (ΔT) of all three samples stabilized at 14.8 K within 60 seconds. This mild temperature rise had a negligible effect on the aptamer, and the temperature on the non-irradiated side remained at 295.15 K. Simultaneously, the ΔV of MXene increased sharply from 0 mV to 23.05 mV; while the ΔV of APT@MXene and APT@MXene+MC-LR increased to 16.08 mV and 21.02 mV, respectively. (t) + (where R represents the proportion of the total current carried by the cations, F is the Faraday constant, and c represents the concentration of the KCl solution in the electrolytic cell), the t of the MXene thin film material can be calculated. + The value is 0.975, APT@MXene is 0.667, APT@MXene+MC-LR is 0.914, and t + The values ​​reflect the changes in K⁺ ion selective transport. Notably, the photothermal-electric response of the aptamer sensor remains stable and repeatable, with the changes in ΔT and ΔV consistently consistent throughout each near-infrared light on-off cycle, demonstrating the excellent cyclic stability of the light-driven MXene nanopore sensor in Embodiment 2 of this invention.

[0064] like Figures 12a-12b The figures show the Zeta potential diagrams of the MXene film, aptamer probe, and MC-LR prepared in the embodiments of the present invention in 0.1 M KCl solution, and the surface charge density diagrams of the MXene film, APT@MXene, and APT@MXene+MC-LR prepared in Example 2 of the present invention. As can be seen from the figures, the Zeta potentials of the MXene film, aptamer probe, and MC-LR are all negative. Therefore, in KCl solution, the negatively charged MXene nanochannels exhibit strong cation selectivity, preferentially adsorbing K+. +Ions simultaneously electrostatically repel Cl - Ions. And from... Figure 12b As can be seen, the surface charge density of the MXene film exhibits a gradual adjustment: the initial measured value is -0.0914 mC / cm. 2 After the aptamer was fixed on the outer surface, the temperature dropped to -0.0532 mC / cm. 2 Subsequently, after aptamer-target binding, the temperature rose to -0.0648 mC / cm. 2 The change in surface charge density is related to the potassium ion selective transport rate calculated above (t). + The changes are consistent with those observed, confirming that negative surface charge enhances cation selectivity. Therefore, this invention concludes that the performance improvement of the light-driven MXene nanopore aptamer electrochemical biosensor originates from the K-induced change in aptamer-target binding. + Changes in ion-selective transport. This effect, dominated by the outer surface charge density, amplifies the current response without aptamer conformational rearrangement, overcoming the fundamental limitation of traditional aptamer sensors that rely on aptamer conformational rearrangement, and achieving higher detection sensitivity.

[0065] Example 6 This embodiment examines the accuracy of the method for detecting microcystin-LR provided by the present invention.

[0066] Table 1 shows the detection of MC-LR concentration in tap water samples using the standard addition method with the light-driven MXene nanopore sensor in Example 2. 1×10⁻⁶ ppm was added to each tap water sample. -3 1×10 -4 1×10 -5 1×10 -6 As shown in the table, the recoveries of MC-LR at four concentrations of spiked tap water samples were all between 104.2% and 109.3%, indicating negligible matrix interference and high quantitative accuracy. Furthermore, the calculated coefficients of variation were all below 4.5%, demonstrating the high reliability of the detection results. This proves that the light-driven MXene nanopore sensor can achieve ultrasensitive MC-LR detection in complex environments.

[0067] Table 1. Accuracy results of the microcystin-LR detection method provided by the present invention.

[0068]

[0069] For any aspects not covered above, existing technologies shall apply.

[0070] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. An electrochemical biosensor with light-driven nanopores, characterized in that, include: The invention comprises a photoresponsive nanoporous film, an aptamer probe, and a near-infrared light source. The photoresponsive nanoporous film is formed by assembling layers of nanosheets, with sub-nanometer spacing between adjacent monolayer nanosheets, constituting sub-nanometer ion channels. The aptamer probe is modified on the outer surface of the photoresponsive nanoporous film for specific recognition of target molecules. Under asymmetric near-infrared light irradiation, the photoresponsive nanoporous film exhibits a temperature gradient through photothermal conversion, thereby promoting the binding of the aptamer probe with the target molecule and causing a change in the charge density on the outer surface of the film, thus enabling the detection of the target molecule.

2. The electrochemical biosensor according to claim 1, characterized in that, The photoresponsive nanoporous film comprises Ti3C2T X Any one of MXene membrane, graphene oxide membrane, carbon nanotube membrane, or porous ionomer membrane with photothermal conversion.

3. The electrochemical biosensor according to claim 1, characterized in that, The photoresponsive nanoporous film is Ti3C2T X MXene membrane.

4. The electrochemical biosensor according to claim 3, characterized in that, The method for preparing the photoresponsive nanoporous thin film includes the following steps: S11. Using HCl / LiF as the etching solution, add the MAX phase Ti3AlC2 and heat and stir the mixture for a period of time. S12. The etching product obtained in step S11 is centrifuged and washed with HCl and deionized water. After the pH value of the supernatant reaches neutral, the supernatant is ultrasonically treated in an inert environment for a period of time. After centrifugation again, the supernatant is taken as the MXene nanosheet colloidal solution. S13. The MXene nanosheet colloidal solution obtained in step S12 is vacuum filtered through a mixed cellulose filter membrane, and then the product is vacuum dried to obtain a photoresponsive nanoporous film.

5. The electrochemical biosensor according to claim 4, characterized in that, The method for modifying the outer surface of the photoresponsive nanoporous film with an aptamer probe specifically includes the following steps: S21. On one side surface of the nanoporous membrane obtained in step S13, a chromium layer and a gold layer are deposited sequentially using a vapor deposition method. S22. The aptamer probe solution is uniformly coated onto the surface of the deposited gold layer and incubated. S23. Wash the unbound aptamer probe with buffer solution and air dry at room temperature to obtain the electrochemical biosensor.

6. The application of the electrochemical biosensor as described in any one of claims 1-5 in the detection of target molecules.

7. The application according to claim 6, characterized in that, The electrochemical biosensor described above is used for the qualitative and quantitative detection of microcystin-LR.

8. A target molecule detection system, characterized in that, The electrochemical biosensor includes any one of claims 1-5.

9. The target molecule detection system according to claim 8, characterized in that, The electrochemical biosensor is placed in a dual-cell electrolytic cell, which is divided into two chambers. Ag / AgCl electrodes are inserted into the two chambers respectively, and KCl solution is added. Near-infrared light is used to irradiate the electrochemical biosensor through the electrolytic cell.

10. The target molecule detection system according to claim 9, characterized in that, The KCl solution concentration is 10. -5 ~10 -1 M.

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